Ab2785

A253 cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized using 0.1% Triton-X-100 (Sigma-Aldrich) for 10 minutes and blocked with 2% bovine serum albumin (BSA) for 30 minutes, all at room temperature. Then, cells were incubated with a mixture of 10 μg/mL mouse anti-galectin-3 antibody (#ab2785, Abcam, USA) in 2% BSA at 4°C overnight. The next day, cells were incubated with a mixture of 10 μg/mL Alexa Fluor-647 anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., USA) in 2% BSA at room temperature for 1 hour.
Formalin-fixed paraffin embedded sections of murine submandibular glands were deparaffinized, rehydrated and subjected to citric acid microwave antigen retrieval. Slides were blocked with 2% BSA (Sigma-Aldrich) and permeabilized by 0.1% Triton-X-100 (Sigma-Aldrich) for 30 minutes at 25°C. Slides were incubated with mouse anti-galectin-3 (#ab2785, Abcam) and rabbit anti-LAMP1 (#21997-1-AP, Proteintech) antibodies at 4°C overnight, followed by incubation with Alexa Fluor-647 anti-mouse IgG and Alexa Fluor-488 anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at room temperature for 1 hour.
Sections were subsequently counterstained with DAPI (#ab104139, Abcam). Images were acquired using a fluorescent microscope (Nikon, Japan) and analyzed using ImageJ software (public domain, source: National Institutes of Health, USA).

Cells were fixed in 4% paraformaldehyde in PBS at room temperature for 15 min, washed and permeabilized with 0.4% Triton X‐100 in PBS for 10 min, and washed again with PBS. Permeabilized cells were blocked with PBS containing 1% BSA for 1 h, and subsequently incubated with GAL‐3 anti‐mouse [1 : 200] (ab2785, Abcam, Cambridge, UK) antibody in blocking buffer overnight at 4 °C (Table S2). Thereafter, cells were washed with PBS and incubated with Alexa‐labeled IgG secondary antibodies containing blocking buffer for 1 h. Slides were incubated with 4′,6‐diamidino‐2‐phenylindole for 3 min, mounted with Fluoromount Aqueous Mounting Medium (Sigma‐Aldrich, St. Louis, MO, USA), and analyzed using a Leica confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA).

Proteins from cells or extracellular vesicles were extracted by lysing the samples in sodium dodecyl sulphate (SDS) loading dye which contains 5 % β-Mercaptoethanol and by subsequent sonication (Bioruptor Plus, Diagenode).
Next samples were subjected to 4–15% SDS-polyacrylamide gel electrophoresis (Biorad). Proteins were transferred by semi-dry electroblotting to PVDF membranes (Biorad). Membranes were exposed to antibodies against Galectin-3 (ab2785, Abcam, 1:1000), total β-Catenin (PA5-19469, Thermo Scientific, 1:200) or GAPDH (MA5-15738, Pierce 1:5000). Anti-Rabbit-IR-Dye 800 (LiCor, 1:10000) or anti-mouse-IR-Dye® 680RD Donkey anti-Mouse (LiCor, 1:10000) were used for subsequent detection by Odyseey (Licor) infrared image system (LiCor).